Introduction

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Steroid action is thought to be mediated by the binding of hormones to specific receptors. Conflicting data, mainly derived from immunohistochemical studies, are available on the presence of estrogen receptors (ERs) in the lung. ERs were not detected in normal tissue, but were either reported to be expressed by a vast majority of lung tumors (1) or were demonstrated in few lung tumors (2). Biochemical assays revealed only 4 of 19 bronchogenic carcinomas to be ER positive (3). Although tamoxifen, a nonsteroidal antiestrogen, is thought to antagonize the effects of estrogens by competing with them for ER binding, it reverses multidrug resistance (4, 5), synergizes with cisplatin cytotoxicity (6), and inhibits growth in ER-negative lung cancer cells (7).

In addition to high-affinity, low-capacity ERs, rat and human target tissues contain a second binding macromolecule termed the type II estrogen binding site (type II EBS). This site shows the same steroid specificity as the ERs, and displays a lower affinity but higher capacity for the ligand than does the ER (8, 9). Because of the relatively low binding affinity (apparent dissociation constant, Kd, ~ 20 nM) of type II EBSs, it is difficult to imagine that these sites could be occupied by estrogens in vivo. A solution to this paradox was suggested by the observation that these sites in rat uterine nuclei are occupied in vivo by a flavonoidlike molecule with growth-inhibitory activity (10, 11).

Flavonoids constitute a widely distributed class of natural substances with a variety of biologic actions (12). It has been found that the flavonoid quercetin binds to type II EBSs present in human mammary (10), ovarian (13), colorectal (14), leukemic (15), and melanoma tumor cells (16), and exerts a powerful inhibitory activity on cell growth, probably by mimicking the endogenous ligand. We have found that tamoxifen can compete with quercetin in whole-cell assays for type II binding, and can inhibit cell proliferation in a dose-dependent manner in a multidrug-resistant, ER-negative MCF-7 breast cancer cell line (17) and in the IM-9 lymphoblastoid cell line, which expresses both ER and type II EBS (18). In this study, we found that type II EBS are expressed in non-small-cell lung cancer cell lines and primary tumors, and that both tamoxifen and quercetin have an antiproliferative effect on these tumor cells. Our data suggest a potential therapeutic role for tamoxifen and quercetin in lung cancers.

	Materials and Methods

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Cell Lines and Tumor Specimens

Human non-small-cell lung cancer cell lines SK-LU1 (adenocarcinoma), SW900 (squamous-cell carcinoma), ChaGo-K-1 (bronchogenic carcinoma), H441 (papillary adenocarcinoma), H661 (large-cell carcinoma), and A549 (adenocarcinoma) were obtained from the American Type Culture Collection (Rockville, MD). Tumor cells were grown as monolayers in 75-cm² T-flasks in RPMI 1640 culture medium (Gibco, Paisley, UK) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gibco) and 2 mM glutamine. Some experiments were also performed with RPMI 1640 medium without phenol red (Eurobio, Les Ulis, France) and with or without charcoal/dextran-treated FCS.

Fresh tissue specimens were collected at the time of surgery, frozen in liquid nitrogen, and stored at 80°C until assay.

Drugs and Chemicals

Quercetin (3,3',4',5,7-pentahydroxyflavone), hesperidin (3',5,3-hydroxy-4-methoxyflavanone), and rutin (the 3-rhamnosylglucoside of quercetin) were purchased from Aldrich (Steinhein, Germany). Steroid hormones were obtained from Sigma (Deisenhofen, Germany). Ipriflavone (isoflavone) and tamoxifen (trans-2[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethyl-ethylamine) were gifts from Chiesi and Zeneca Pharmaceuticals (Italy), respectively.

Growth Experiments

Cells were plated at 10⁴/cm² in 16-mm wells of a 24-well plate (Falcon; Becton Dickinson, Oxnard, CA). After 18 h, the medium was replaced with fresh medium containing the compounds to be tested. The compounds were added from an absolute ethanol stock solution, and the control cells were treated with the same amount of vehicle alone. The final ethanol concentration did not exceed 0.5% (vol/ vol) either in control or in treated samples. The treatments were repeated after 24 h. Quadruplicate hemocytometer counts of triplicate cultures were performed after 48 h of exposure to the compounds. To determine the level of thymidine incorporation, cells were plated at 10⁴/cm² in 96-well microtiter plates (Falcon) in 10% medium. After 18 h the medium was removed and the cells were maintained in 0.2% FCS medium for 72 h to induce cell-cycle arrest. The medium was then replaced with fresh 10% FCS medium containing the compounds to be tested or vehicle, as described earlier. Thymidine incorporation was assayed at 2, 24, 48, 72, and 96 h. Cells were incubated with 1 µCi/well of [³H]thymidine deoxyriboside ([³H]TdR) (specific activity: 5 Ci/mmol; Amersham, Little Chalfont, UK) for 2 h. Cells were harvested with a cell harvester (Inotech, Dotticon, Switzerland) on a glass microfiber filter (Whatman, Maidstone, UK), and the level of [³H]TdR incorporation into DNA was evaluated with an automated filter counting system (Inotech) by measurement of ionizing radiation.

Estrogen Receptor (ER) Analysis

Cells in 24-well plates (4 to 8 × 10⁴ cells/well), in RPMI 1640 medium supplemented with 20 mM 4-(2-hydroxyethyl)-1-piperazine-N'-2 ethanesulfonic acid (HEPES) without serum, were incubated with increasing concentrations (0.1 to 2.0 nM) of 2,4,6,7-³H-estradiol (Amersham; specific activity: 85 to 110 Ci/mmol) alone or in the presence of a 100-fold excess of unlabeled diethylstilbestrol for 30 min at 37°C in a 5% CO₂-air atmosphere. Preliminary experiments showed that this time interval exceeded that required for the achievement of maximal binding. Specific binding was calculated as the difference between total binding (wells without competitive binding) and nonspecific binding (wells with competitive binding). The concentration and apparent equilibrium dissociation constant (Kd) of receptor sites were obtained by Scatchard analysis. The conversion of radioactivity (dpm) to number of sites per cell was accomplished by determining the amount of [³H]estradiol bound for aliquots derived from a known number of cells, and by then applying Avogadro's number. ERs in primary tumor specimens were assayed with the dextran-coated charcoal (DCC) method, as previously described (19). The observed number of binding sites was expressed as femtomoles of [³H]ligand specifically bound per mg of cytosolic protein.

Type II EBS Analysis

Cells were analyzed for type II EBS with a whole-cell assay, as previously described (14), with slight modifications. Briefly, 2 to 5 × 10⁴ cells/well in 24-well plates in medium with 20 mM HEPES, without serum, were incubated at 4°C for 2.5 h with increasing concentrations (4 to 60 nM) of 6,7-[³H]estradiol (Amersham; specific activity: 40 to 60 Ci/mmol) alone or in the presence of a 200-fold molar excess of unlabeled diethylstilbestrol. The cells were washed twice with ice-cold medium and then resuspended in 0.8 ml 80% (vol/vol) absolute ethanol to extract bound steroid. Specific binding was calculated as the difference between total and nonspecific binding. The results were expressed as the number of binding sites per cell. Cytosolic type II EBSs in tumor specimens were assayed by the hydroxyapatite method, as previously described (14). To determine the effect of sulfydryl reduction on type II EBS, the cytosol was preincubated with 10 mM dithiothreitol for 1 h at 4°C. Nuclear type II EBS was assayed according to the procedure developed by Syne and colleagues (9), with minor modifications. Briefly, the tissue was homogenized (200 to 500 mg/ml) in TE buffer (10 mM Tris, 1.5 mM ethylene diamine tetraacetic acid [EDTA], pH 7.4) at 4°C, and the homogenate was centrifuged at 2,000 × g for 30 min. The pellet was then resuspended in the same buffer and used for the sucrose pad nuclear exchange assay. One-milliliter aliquots of homogenate were layered onto 1-ml pads of 1.2 M sucrose and centrifuged in a swinging-bucket rotor at 7,000 × g for 45 min at 4°C. The supernatant and the sucrose pad were discarded and the nuclear pellet was incubated in the presence of increasing concentrations (4 to 70 nM) of [³H]estradiol (Amersham; specific activity: 40 to 60 Ci/mmol) alone or in the presence of a 300-fold molar excess of unlabeled diethylstilbestrol for 60 min at 30°C. At the end of the incubation period, the tubes were chilled on ice for 5 min. Electron microscopic observations of the nuclear preparations did not reveal any contaminating rim of cytoplasmic material around the nuclei. Receptor-steroid complexes were then separated from free steroid by precipitation with cold protamine sulfate (1 mg/ml) in TE buffer for 10 min at 4°C. The samples were then diluted with 6 ml of TE buffer containing 1% Tween 80 (Sigma, Deisenhofen, Germany) and centrifuged at 2,000 × g for 10 min. The resulting pellets were washed once with 6 ml of the same buffer and then extracted overnight with 1 ml of 80% ethanol. Radioactivity was measured by liquid scintillation spectrometry. Specific binding was calculated as the difference between binding in the absence (total binding) and in the presence (nonspecific binding) of diethylstilbestrol, and was reported as bound fmol/mg of nuclear DNA. The DNA concentration in nuclear preparations was assayed with the method described by Burton (20).

Growth Experiments on Primary Tumors

The effect of tamoxifen, quercetin, rutin, ipriflavone, and hesperidin on cancer cell growth in tumor specimens was assessed in vitro with the Amersham cell proliferation kit according to the manufacturer's protocol. Briefly, tumor slices were incubated in sealed tubes at 37°C in Dulbecco's modified Eagle's medium containing 20 mM N-2-ethanesulfonic acid (Gibco) and 10% FCS, in the presence of the compound to be tested (10 µM) or vehicle alone (0.5% absolute ethanol). After the 3-h incubation, 100 µM 5-bromo-2'-deoxyuridine (BrdU), 10 µM 5-fluoro-2'-deoxyuridine (to block intracellular thymidilate synthase activity), and 0.3% (vol/vol) H₂O₂ were added to the incubation medium and the resealed tubes were incubated for an additional 2 h at 37°C. Slices from the same tumor were immediately pulsed for 2 h with BrdU without previous incubation with quercetin or vehicle alone, in order to assess the effect of this preincubation time interval on the BrdU labeling index of the tumor. The tissue slices were then washed in phosphate-buffered saline (PBS) at 37°C for 15 min and fixed in 10% neutral-buffered formalin for 12 h. Standard dehydration and paraffin wax-embedding procedures were used on fixed tissue. After 1 h incubation at room temperature with the anti-BrdU antibody and nuclease mixture (Amersham), the tissue slices were treated with the peroxidase-labeled secondary antibody and the reaction product was revealed by exposure to 3,3'-diaminobenzidine tetrahydrochloride. The percentage of BrdU-labeled cells, both in control and quercetin-treated samples, was measured in a count of 1,500 neoplastic cells identified by histologic criteria. The proportion of proliferating-cell nuclear antigen (PCNA)-positive cells was evaluated immunohistochemically by using an anti-PCNA monoclonal antibody (clone PC10, 1:100; Ylem, Avezzano, Italy). The percentage of PCNA-positive cells was evaluated in the same way for the BrdU assay.

	Results

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Lung Carcinoma Cell Lines Express ER and Type II EBS

Evidence of ER and type II EBS binding of [³H]estradiol in SK-LU1, SW900, ChaGo-K-1, H441, A549, and H661 cell lines was observed by saturation analysis over a broad range of concentrations in whole-cell assays. The [³H]estradiol saturation analysis done on ChaGo-K-1 cells at 4°C for 2.5 h resulted in a sigmoid curve, with saturation occurring at [³H]estradiol concentrations of approximately 30 nM (Figure 1A). As predicted by the biphasic nature of the saturation curve, Scatchard analysis of the binding data yielded a concave plot (Figure 1B) similar to that observed in other tissues (9, 14). Because an accurate estimate of both the Kd value and the number of type II EBSs cannot be made from a curvilinear Scatchard plot, these parameters were obtained from the saturation curve. For the experiment shown in Figure 1, the number of type II EBSs in ChaGo-K-1 cells, calculated at maximal binding, was 61.9 × 10⁴ sites/cell. The corresponding Kd value, determined from the [³H]estradiol concentration required for half-maximal saturation, was about 15 nM. Hill analysis of these data yielded a Hill coefficient greater than 2 (data not shown). Although the results are not conclusive, the concave Scatchard plot and Hill coefficient for these sites suggest that they are multiple and display positive cooperativity. The results of four similar experiments performed on SK-LU1, SW900, H441, A549, and H661 cell lines are shown in Table 1.

View larger version (18K): [in this window] [in a new window]   Figure 1.   Expression of type II EBS by ChaGo-K-1 lung carcinoma cell line. Cells were analyzed for type II EBS with a whole-cell assay. Cells were incubated at 4°C for 2.5 h at increasing concentrations (4 to 70 nM) of 6,7-[3H]estradiol (specific activity: 40 to 60 Ci/mmol) alone or in the presence of a 200-fold molar excess of unlabeled diethylstilbestrol to determine total (squares) and nonspecific (triangles) binding, respectively. Specific binding (circles) was calculated as the difference between total and nonspecific binding. (A) Saturation analysis. (B) Scatchard plot of data in (A).

To avoid the interference of type II EBSs in ER measurement, and to allow [³H]estradiol exchange with occupied ERs, we performed titration analysis of ERs in separate experiments at 37°C for 30 min with [³H]estradiol concentrations in the range of 0.1 to 2.0 nM. All cell lines expressed ERs with Kd values ranging from 0.2 to 0.6 nM (Table 1).

Steroid-specificity experiments demonstrated that ERs and type II EBSs in lung carcinoma cell lines are estrogen-specific. Only those steroids with estrogenic activity inhibited binding of [³H]estradiol to ERs and type II EBSs (Table 2). Both tamoxifen and quercetin were able to compete with [³H]estradiol for type II EBSs, with a potency similar to that of diethylstilbestrol. Contrastingly, the 3-rhamnosylglucoside of quercetin, rutin, and the isoflavone ipriflavone did not compete for type II EBS binding. As expected, the antiestrogen tamoxifen interacted with ERs, whereas quercetin, rutin, and ipriflavone did not.

Tamoxifen and Quercetin Inhibit the Growth of Lung Carcinoma Cell Lines

Because lung carcinoma cells contain type II EBSs, it seemed possible that they could be sensitive to quercetin. Indeed, not only quercetin but also tamoxifen at a concentration between 10⁸ and 10⁶ M inhibited the proliferative activity of these cells in a concentration-dependent manner (Figure 2). In addition, the magnitude of the growth-inhibitory effect of tamoxifen, quercetin, and diethylstilbestrol (data not shown) was similar. However the cell lines tested varied in their sensitivity to the inhibitory effect of tamoxifen and quercetin. H661 cells were less sensitive, since their growth was inhibited by 50% by tamoxifen and quercetin concentrations of 1.72 µM and 2.28 µM, respectively (Table 3). Rutin and ipriflavone, which did not bind to type II EBSs, were ineffective in inhibiting cell growth.

View larger version (19K): [in this window] [in a new window]   Figure 2.   Quercetin and tamoxifen inhibit the growth of lung carcinoma. SK-LU1 cells were plated at 104/cm2 in 16-mm wells of a 24-well plate, and the medium was replaced after 18 h with fresh medium and the compounds added at the indicated concentrations. The treatments were repeated after 24 h. Quadruplicate hemocytometer counts of triplicate cultures were performed after 48 h. The control value was 82,333 ± 3,416 cells/well. Open triangles, rutin; open squares, ipriflavone; closed triangles, quercetin; closed circles, tamoxifen. Results represent the mean ± SD of four experiments.

Despite a 20% reduction of the cell number at the end of the incubation period in cultures supplemented with charcoal/dextran-stripped FCS, no modifications of the activity of the various molecules tested were observed. On the other hand, the growth pattern of tumor cells was not modified by removing phenol red from the medium (21), and diethylstilbestrol, at concentrations able to saturate ERs, had no effect on plating efficiency or proliferation of cell lines. We observed that addition of 10¹⁰, 10⁹, and 10⁸ M diethylstilbestrol to cultures containing charcoal/ dextran-stripped FCS in the absence of phenol red did not modify cell proliferation, whereas higher concentrations, such as 10⁷, 10⁶, and 0.25 × 10⁵ M diethylstilbestrol, were inhibitory in a dose-dependent manner. Moreover, the inhibitory effect of tamoxifen and quercetin could not be reversed by the addition of exogenous estrogens (data not shown). The inhibitory effect of tamoxifen, quercetin, and diethylstilbestrol was not attributable, in the range of concentrations used, to a cell-killing action of these substances. In fact, cell viability after 3 days' culture did not differ between control and treated cells, being more than 85%. Furthermore, the growth-inhibitory effect appeared to be reversible, since after the removal of tamoxifen, quercetin, or diethylstilbestrol from the culture medium, the treated cells grew again like untreated cells (Figure 3).

View larger version (23K): [in this window] [in a new window]   Figure 3.   Reversibility of the antiproliferative effect of quercetin, tamoxifen, and diethylstilbestrol on SK-LU1 cells. Cells were plated at 104/cm2 in 16-mm wells of a 24-well plate, and after 18 h (0 h) the medium was replaced with fresh medium containing 1 × 105 M quercetin (triangles), 0.25 × 105 M tamoxifen (circles), 0.25 × 105 M diethylstilbestrol (squares), or vehicle alone (0.5% absolute ethanol) (crosses). After 24 h, the medium in the quercetin-, tamoxifen-, and diethylstilbestrol-treated cultures was replaced with fresh medium containing vehicle alone or the corresponding compounds (dotted lines). Results represent the means of quadruplicate determinations, at the indicated times, from one of three experiments. SDs of means were less than 10% and were omitted from the figure.

We also evaluated the effects of tamoxifen and quercetin on DNA synthesis. Both compounds, at doses that clearly inhibited cell growth, were able to inhibit thymidine incorporation in a time-dependent manner; these effects were evident 2 h after addition of the compounds (Figure 4).

View larger version (26K): [in this window] [in a new window]   Figure 4.   Quercetin and tamoxifen inhibit DNA synthesis. H441 cells were plated at 104/cm2 in a 96-well plate and after 18 h the medium was removed and the cells maintained in 0.2% FCS medium for 72 h to induce cell-cycle arrest. The medium was then replaced with fresh 10% FCS medium containing the compounds to be tested. [3H]thymidine incorporation was assayed at the indicated times. The control values (cpm) were 2 h = 2,690 ± 105; 24 h = 11,725 ± 483; 48 h = 13,195 ± 339; 72 h = 15,162 ± 423; 96 h = 16,563 ± 408. Open squares, 1 × 105 M rutin; open circles, 1 × 105 M ipriflavone; closed triangles, 0.25 × 105 M quercetin; closed circles, 0.5 × 105 M quercetin; closed squares, 1 × 105 M quercetin; open triangles, 0.25 × 105 M tamoxifen. Results represent the mean ± SD of four experiments.

Primary Tumors Express Type II EBSs

Tumor specimens from patients with lung tumors were evaluated for the presence of type II EBS and ER (Table 4). Cytosolic and nuclear type II EBSs were assayed with [³H]estradiol concentrations ranging fron 4 to 70 nM, and were expressed in appreciable amounts in all cases studied. The range of receptor concentrations was from 680 to 5,670 fmol/mg of protein, with Kd values ranging from 15 to 25 nM, for cytosolic receptors, and from 380 to 11,680 fmol/mg of DNA, with Kd values ranging from 12 to 22 nM, for nuclear receptors. The shape of both the binding curve of [³H]estradiol to type II EBSs and the Scatchard plot of binding data closely resembled those observed for cell lines (data not shown). In the presence of 10 mM dithiothreitol (DTT), the binding of [³H]estradiol to type II EBSs was reduced to approximately 30% of the control (without DTT) value (data not shown). This sensitivity is similar to that previously observed for type II EBSs in rat uterus (8), human colorectal (19) and ovarian cancers (13), and melanoma (16).

As in lung carcinoma cell lines, tamoxifen and quercetin displaced [³H]estradiol from cytosolic type II EBSs in primary tumor samples with a potency similar to that of diethylstilbestrol, whereas nonestrogenic steroids, as well as rutin, ipriflavone, and hesperidin, did not (Table 5). Similar results were obtained for nuclear type II EBSs (data not shown).

High-affinity, low-capacity ERs were assayed with [³H]estradiol concentrations ranging fron 0.1 to 2.0 nM. Seven of the 14 cases examined expressed low amounts of ERs with Kd values and steroid specificities (data not shown) similar to those described for ERs in classic target organs (Table 4). ER-negative cases included squamous cell tumors and a poorly differentiated adenocarcinoma (patient P.M.).

Growth Inhibition in Primary Tumors

To evaluate the tamoxifen and quercetin sensitivity of proliferating tumor cells in primary tumors, we used measurement of BrdU uptake. Because we had observed that in the cell lines the inhibitory effects of quercetin and tamoxifen became evident after 2 h of treatment, we pretreated tumor specimens in vitro for 3 h before pulsing with BrdU. As shown in Figures 5A and 5B and in Table 6, the number of BrdU-labeled nuclei was greatly reduced in tamoxifen- and quercetin-treated specimens as compared with untreated ones. Similar treatment with rutin, ipriflavone, or hesperidin did not reduce the percentage of labeled cells. Similar results were also obtained by assessing the number of PCNA-positive cells (Figures 5C and 5D and Table 6).

View larger version (154K): [in this window] [in a new window]   Figure 5.   Slices of tumor from patient S.G. (a and b) and patient P.A. (c and d) were incubated for 3 h in the absence (a and c) or presence (b and d) of 10 µM quercetin. Lung carcinoma cells labeled with BrdU were visualized by labeling with a monoclonal anti-BrdU antibody (a and b). In c and d, cells were stained with a monoclonal antibody against PCNA. Bars = 35 µm.

	Discussion

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Non-small-cell lung carcinomas specifically bind estrogen. This binding is mainly due to the presence of large numbers of type II EBSs, whereas ERs are absent or present at low concentrations. Moreover, the presence of this second binding site can interfere with the accurate quantitation of ERs. This is particularly true when an inappropriate range of tracer concentrations is used to assay ERs (i.e., at concentrations higher than 3 nM, the tritiated estradiol tracer, in addition to binding to ERs, also binds to type II EBSs).

The concave Scatchard plot and the Hill coefficient of more than 2 for type II EBSs suggest that they are multiple and display positive cooperation. Another distinguishing characteristic of type II EBS binding is its sensitivity to the presence of sulfhydryl-reducing reagents such as DTT (8), shown in lung carcinomas.

Tamoxifen and quercetin can compete for binding to type II EBSs with a potency similar to that of estrogens. On the other hand, rutin, the rhamnosylglucoside of quercetin, ipriflavone, and hesperidin do not compete with estrogens for these sites.

In addition to high concentrations of type II EBSs, appreciable amounts of ERs are also expressed by lung carcinoma cell lines, and as expected (22), estrogens and the antiestrogen tamoxifen interact with these receptors, whereas quercetin, rutin, and ipriflavone do not.

As also shown in other cell types (16, 23), despite the presence of ER, neither estrogens nor antiestrogens, at concentrations able to saturate these receptors, had any effect on plating efficiency or proliferation of lung carcinoma cell lines (data not shown).

Only those substances (tamoxifen and quercetin, but not ipriflavone and rutin) that can interact with type II EBSs exert a dose-dependent growth-inhibitory effect on lung carcinoma cell lines. In addition, tamoxifen and quercetin, but not ipriflavone, rutin, or hesperidin, inhibit the in vitro growth of cells from primary tumors. This is in agreement with previous studies showing that type II EBSs mediate the inhibitory effect of both quercetin and other flavonols (10, 13, 15) and tamoxifen (16) in human cancer cell lines and primary tumors.

In an ER-negative lung carcinoma cell line, in vitro exposure to pharmacologic concentrations of tamoxifen results in growth inhibition (7). This ER-independent inhibitory activity of tamoxifen and related triphenylethylenes is supported by several other lines of evidence. These include the tamoxifen-induced growth inhibition of some ER-negative cell lines (17, 24) and of ER-positive cancer cell lines maintained under serum-free conditions (i.e., in the absence of exogenous estrogens), as shown in both the present study and other studies (16, 25), and the clinical response to tamoxifen of a distinct proportion of patients with ER-negative breast tumors (22). Moreover, the inhibitory effects of high tamoxifen concentrations on ER-positive cells cannot be reversed by the addition of exogenous estrogens, and these inhibitory effects are not related to the affinity of this antiestrogen for ER, as also shown in our study and another (26).

A basis for the ER-independent growth-inhibitory effects of tamoxifen and other related compounds has recently been suggested. Several groups have shown that these compounds are potent inhibitors of protein kinase C (PKC) (25, 26), which is known to play a role in the signal-induced cascade involved in regulating proliferation in many cell types. However, the 50% inhibitory concentrations for PKC activity recorded for tamoxifen and chemically related compounds were in the range of 15 to 100 µM (27), which is far from those which we observed to be effective in inhibiting cell growth. Flavonoids interact with a variety of enzymes including PKC (28). Although it is possible that they may act at the levels described for tamoxifen and related compounds, the effective concentrations of the interactions involving flavonoids are in the micromolar (~ 50 µM) range. Conversely, both tamoxifen and quercetin interact with type II EBS and inhibit the growth of lung carcinoma and other neoplastic cells at lower concentrations (13). It therefore seems likely that the growth-inhibitory effect of tamoxifen and quercetin is mediated, as in other systems, by their interaction with type II EBS (10, 11, 13).

A common mechanism of action for these compounds is further suggested by several similar effects that they produce in different tumor cell types, including lung cancer cells. Among them are the capacity: (1) to induce apoptosis (29, 30); (2) to reverse the phenotype of multidrug resistance (17); and (3) to interact in a highly synergistic manner with cisplatin both in vitro and in vivo (6, 31).

Then again, the presence of type II EBS in cell lines and in specimens from patients suggests a potential role for tamoxifen and quercetin in the treatment of lung tumors. The in vitro inhibitory effect of tamoxifen and its synergistic interaction with other drugs are compatible with the concentrations that can be obtained in human plasma (35). In this context, it is also to be noted that a plasma concentration of 12 µM quercetin, which is higher than that effective in vitro for inhibiting lung carcinoma cell proliferation, was obtained after an intravenous injection of 100 mg without any apparent side effects (36).

The observation that a dietary supplement of quercetin inhibits the development of 7,12-dimethylbenzoanthracene- and N-nitrosomethylurea-induced rat mammary cancer (37) strongly supports the possibility that quercetin could also be active in vivo. The growth-regulatory role of quercetin might partially explain the correlation between high dietary intake of flavonoids in humans and a lower incidence of cancer of the stomach, colon, and breast (38) in populations with such an intake. In addition, it has been recently shown that chalcones (open-chain flavonoids) inhibit pulmonary carcinogenesis in mice (41), and a Phase I clinical trial has shown evidence of an antitumoral activity of quercetin in a series of solid tumors, including non-small-cell lung cancer (42).

The mechanisms by which tamoxifen and quercetin  inhibit cell growth after ligand-receptor interactions are unclear. These substances can induce the production of transforming growth factor- (TGF-) and their growth- inhibitory effect is attributable to a TGF--mediated autocrine loop in leukemic, breast, and ovarian neoplastic cells (43). Our preliminary observations suggest that tamoxifen and quercetin induce TGF-₁ production in lung cancer cell lines. TGF-₁ can inhibit lung carcinoma cell growth (46, 47), and induction of TGF-₁ may therefore be one of the mechanisms by which tamoxifen and quercetin inhibit lung cancer cell growth.